I propose to investigate the molecular mechanisms of regulation and calcium transport in molluscan muscles using combined biochemical and structural approaches involving protein purification and characterization, electron microscopy and image analysis. Control of muscle contraction is mediated by specific regulatory proteins within the thin and/or thick filaments. These proteins are switched on by cytoplasmic messengers (such as Ca2+ and cyclic nucleotides), which act either directly on the regulatory proteins or through other protein intermediates. I propose to study the role of phosphorylation in the regulatory mechanism of molluscan smooth muscles. Many of these muscles are adapted for prolonged tonic contraction -- "catch"; the ability of maintaining tension economically for long periods -- which is found to a lesser degree in smooth muscles of vertebrates ("latch"). In skinned molluscan fibers the catch state can be relaxed by cyclic nucleotides without any change in the Ca2+ concentration. I plan to exploit this unusual property by investigating the phosphorylation of specific muscle proteins in relation to the state of contraction of the whole muscle monitored by tension measurements. Preliminary experiments indicated that the rod portion of myosin can be phosphorylated in vitro by an endogenous heavy chain kinase. Using isolated proteins I plan to quantify and localize the phosphorylation sites on the purified myosin. I will also carry out structural studies by electron microscopy to analyze the effect of phosphorylation on the conformation and self-assembly of the myosin molecules. Together, these studies may be critical in understanding the assembly process of myosin into filaments and may reveal how the prolonged tonic state is controlled in catch muscles. The sarcoplasmic reticulum membrane system is an integral part of the complex switching mechanism that controls muscle contraction. I plan to study the effect of calcium on the highly ordered structure of molluscan sarcoplasmic reticulum, both in situ by sectioning and freeze-fracture techniques, and in isolated vesicles by negatively staining for electron microscopy. I will also examine the structural dynamics of this system by controlled cross-linking of the Ca2+ pumping protein in the membrane and by visualizing the structure in different "trapped" physiological states using electron microscopy. Determining the dynamical arrangement of the Ca2+ pump is critical to understanding the functional role of the SR membrane system in a variety of muscle types.